Site directed mutagenesis kit

To create the GLP1R 3′-UTR luciferase reporter plasmid, a pMIR-REPORT vector (Ambion, USA) was used, and nucleotides 262–461 of the GLP1R 3′-UTR were cloned into the plasmid. A similar plasmid with a mutated miR-192 seed sequence (from AGGTCAA to CATGTGC) was also constructed using a site-directed mutagenesis kit (TianGen, China).
HK-2 cells were seeded in a 24-well plate and transfected with a mixture of 1 µg of pMIR-REPORT plasmid, 0.2 µg of β-gal plasmid (Ambion, USA), and 50 nmol miR-192 or negative control (NC) mimic and inhibitor with Lipofectamine 3000 (Invitrogen, CA, USA). The cells were lysed 48 h after transfection, and luciferase activity was measured with a luciferase assay kit (Beyotime, China). All experiments were repeated three times.

The genes encoding the RBP and RBP variants were cloned into expression vectors by PCR followed by restriction enzyme digestion and ligation. All RBP variants were obtained using a site-directed mutagenesis kit (TIANGEN). All sequences were confirmed by DNA sequencing. See the SI for experimental details.

Genes were heterologously expressed in Escherichia coli BL21(DE3) harboring the plasmid pET28a. The mutants were created using the site-directed mutagenesis kit from Tiangen Biotech. The primer pairs used for site-directed mutagenesis are presented in Supplementary Table S1. E. coli BL21(DE3) was grown in lysogeny broth at 37°C with 200 rpm shaking until the optical density (OD₆₀₀) of the cultures reached 0.8. The cultures were then cooled to 16°C and isopropyl β-d-1-thiogalactopyranoside was added to a finial concentration of 0.5 mM. The cells were further incubated at 16°C with 200 rpm shaking for 20 h. The cultures were centrifuged at 12 000 g for 10 min at 4°C. The cell pellets were resuspended in 50 mM Tris–HCl buffer (pH 8.0, 150 mM NaCl). The suspension was subjected to ultrasonication to disrupt the cells. The cell lysate was then centrifuged at 12 000 g for 20 min at 4°C. The supernatants were filtered through a 0.45-μm membrane (Merck Millipore) and then loaded onto AKTA pure (Cytiva, MA, USA) coupled with a HisTrap HP column (5 ml, Cytiva). The product fraction was eluted with imidazole-containing (50–500 mM) elution buffer (10 mM Tris–HCl, 150 mM NaCl, pH 8.0) at a flow rate of 1 ml/min. The collected product fractions were further subjected to a desalting column (5 ml, Cytiva) with elution buffer to remove imidazole.

StarBase2.0 was used to predict the potential binding sites of miRNAs on DSCAM-AS1 [32 (link)]. Generation of Wild-type (WT) DSCAM-AS1 with potential miR-384 binding sites were inserted into a luciferase reporter vector psi-CHECK-2 (Promega, USA). In addition, a site-directed mutagenesis kit (Tiangen, Beijing, China) was used to generate a mutated form of this vector termed MT-DSCAM-AS1.Co-transfection of CRC cells with luciferase plasmids and miR-384 mimics or miR-NC were carried out followed by culturing for 48 h. Dual-Luciferase Reporter Assay System (Promega) was used to determine the luciferase activities, following the manufacturer’s instructions.